The present invention relates to a novel piperidine derivative represented by the following formula (1) which shows an inhibitory activity against farnesyl transferase: 
in which A, E and G are defined as described below, or pharmaceutically acceptable salts thereof.
The present invention also relates to a process for preparation of the compound of formula (1), to intermediates which are used in the preparation of the compound of formula (1), and to a pharmaceutical composition comprising the compound of formula (1) as an active ingredient.
Mammalian Ras proteins act as molecular switches in the signalling events associated with cell growth and differentiation. The ras proto-oncogene family consists of three members, N-, K-, and H-ras, which code for highly homologous 4 types of proteins; i.e., H, N-ras proteins of 189 residues and two isomorphic K-ras-4B and K-ras-4A proteins of 188 and 189 residues, respectively. The chemical basis for the switch mechanism involves cycling of the protein between the inactive (off) guanosine diphosphate (GDP) bound state and the active (on) guanosine triphosphate (GTP) bound state (Bourne, H. R.; Sanders, D. A.; McCormick. F.; Nature, 1991, 349, 117). Biochemical and structural studies have shown that point mutations of the residues 12, 13 and 61, positioned in the neighborhood of phosphoryl ground of GTP, resulting in the decrease of guanosine triphosphatase activity are associated with many human cancers, particularly, pancreatic cancer, urinary bladder carcinoma, colon cancer, etc. (Bos, J. L., Cancer Res., 1989, 49, 4682).
Ras protein is synthesized as a cytosolic precursor that ultimately localized to the cytoplasmic face of the plasma membrane after a series of posttranslational modification (Gibbs, J. B., Cell 1991, 65, 1). These series of biochemical modifications, by changing the electrical charge state or spacial structure to increase the hydrophobicity allow Ras protein to attach to cell membrane more easily. The first and obligatory step in the series is the addition of a farnesyl moiety to the cysteine residue of the C-terminal CAAX motif (C, cysteine; A, usually aliphatic residue; X, any other amino acid) in a reaction catalyzed by farnesyl protein transferase (FTase). This modification is essential for Ras function, as demonstrated by the inability of Ras mutants lacking the C-terminal cysteine to be farnesylated, to localize to the plasma, and to transform mammalian cells in culture (Hancock, J. F., Magee, A. I., Childs, J. E., Marshall, C. J., Cell 1989, 57, 1167). The subsequent posttranslational modifications, cleavage of the AAX residues, carboxyl methylation of the the farnesylated cysteine, and palmitoylation of the cysteines located upstream of the CAAX motif in H- and N-ras proteins are not obligatory for Ras membrane. association or cellular transforming activity. Interestingly, K-ras-4B, different from H- and N-ras, has a multiple lysine rich region named polybasic domain, instead of having cysteine required for palmitoylation, thereby facilitating the farnesylated ras protein to bind to anionic lipid layer of cell membrane. The inhibitors of FTase that catalyzes the obligatory modification have therefore been suggested as anticancer agents for tumors in which Ras oncogene contributes to transformation (Buses, J. E. et al., Chemistry and Biology, 1995, 2, 787). A number of FTase inhibitors recently identified demonstrated potent and specific ability to block Ras farnesylation, signalling and transformation in transformed cells and tumor cell lines both in vitro and in animal models (Kohl. N. E. et. al., Proc. Natl. Acad. Sci. USA. 1994, 91, 9141; Kohl, N. E. et al., Nature Medicine, 1995, 1 792).
However, most of the inhibitors are related to CAAX motif as Ras substrate mimic and peptidic in nature or contain a sulfhydryl group (U.S. Pat. No. 5,141,851; Kohl, N. E. et. al., Science, 1993, 260, 1934; PCT/US95/12224, Graham et al.; Sebti S. M. et. al., J. Biol. Chem., 1995. 270, 26802; James, G. L. et al., Science, 1993, 260, 1937; Bishop, W. R. et al., J. Biol. Chem., 1995, 270, 30611). Recently, a new type of peptidomimetic inhibitor imitating catalytic step of FTase has been reported (Poulter, C. D. et al., J. Am. Chem. Soc., 1996, 118, 8761). The chemical basis of the inhibitor design relates to the reaction mechanism. This is, transferring prenyl group by the enzyme is electrophilic displacement and the reaction requires (+) charge in a transition state.
These inhibitors previously described however possess limited activity and selectivity for inhibition of the oncogenic function of Ras proteins, particularly K-ras-4B, which is found to be most common in human cancer. Therefore, new inhibitor having the ability of effectively inhibiting K-ras activity is required.
With regard to the restenosis and vascular proliferative diseases, it has been shown that inhibition of cellular ras prevents smooth muscle proliferation after vascular injury in vivo (Indolfi C. et al., Nature Med., 1995, 1(6), 541-545). This report definitively supports a role for farnesyl transferase inhibitors in this disease, showing inhibition of accumulation and proliferation of vascular smooth muscle.
The present inventors have performed studies for developing a compound having the structure characteristics imitating an intermediate state of catalytic reaction of FTase and as a result, found that piperidine derivatives according to the present invention can potently inhibit the enzyme.
Therefore, the object of the present invention is to provide a piperidine derivative of formula (1) which inhibits the activity of FTase, process for preparation thereof, and a novel intermediate which can be used effectively in the process for preparing the compound of formula (1).
It is another object of the present invention to provide a pharmaceutical composition comprising the compound of formula (1) as an active ingredient.
It is the first object of the present invention to provide a piperidine derivative represented by the following formula (1) and pharmaceutically acceptable salts thereof which inhibit the activity of farnesyl transferase: 
in which
A represents hydrogen, lower alkyl, or 
xe2x80x83wherein
B represents CH2, Cxe2x95x90O or SO2, and
D represents a radical selected from the following group: 
xe2x80x83In the definition for the substituent D,
m denotes an integer of 0 to 3,
n denotes an integer of 1 to 3,
X represents hydrogen, phenyl, phenoxy, lower alkyl, lower alkoxy, halogen, nitro, or amino which is optionally substituted by benzyl or lower alkyl,
R1 and R2 independently of one another represent hydrogen, lower alkyl, C3-C6-cycloalkyl, lower alkyl substituted by C3-C6-cycloalkyl, aryl or heteroaryl,
E represents hydrogen, phenyl, naphthyl or 
xe2x80x83wherein
R3 and R4 independently of one another represent hydrogen, lower alkyl, aryl or 
xe2x80x83(wherein Y represents O or S, nxe2x80x2 denotes an integer of 2 to 4, and R5 represents lower alkyl),
G represents a radical selected from the following group: 
xe2x80x83wherein
Z represents O, S, SO2 or Nxe2x80x94R6 (wherein R6 represents hydrogen or lower alkyl),
R7 and R8 independently of one another represent hydrogen, lower alkyl, lower alkoxy, halogen, cyano, hydroxycarbonyl, aminocarbonyl, aminothiocarbonyl, hydroxy, phenyl or phenoxy.
Particularly, the compound according to the present invention has a quite different structure from the known inhibitors for farnesyl transferase, and furthermore it does never include the thiol moiety.
In the definitions for the substituents of the compound of formula (1), the term xe2x80x9clower alkylxe2x80x9d means a straight-chain or branched alkyl having 1 to 4 carbon atoms which includes methyl, ethyl, isopropyl, isobutyl and t-butyl; the term xe2x80x9ccycloalkylxe2x80x9d means cyclic alkyl which includes cyclohexyl; the term xe2x80x9carylxe2x80x9d means 6 to 14-membered monocyclic-, bicyclic- or tricyclic aromatic group; and the term xe2x80x9cheteroarylxe2x80x9d means 6 to 14-membered monocyclic-, bicyclic- or tricyclic aromatic group containing hetero atom(s) selected from a group consisting of oxygen, nitrogen and sulfur.
Also, the compound of formula (1) according to the present invention can form a pharmaceutically acceptable salt. Such salt includes non-toxic acid addition salt containing pharmaceutically acceptable anion, for example a salt with inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydriodic acid, etc., a salt with organic carboxylic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, trofluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, asparagic acid, etc., or a salt with sulfonic acids such as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, etc.; base addition salt for example a salt with pyridine or ammonia; and metal addition salt for example a salt with alkali metal or alkaline earth metal such as lithium salt. Further, the present invention includes a solvate of the compound of formula (1) such as alcoholate or hydrate thereof. They can be produced by conventional conversion methods.
Among the compound of formula (1) according to the present invention, the preferred compounds include those wherein
A represents hydrogen, lower alkyl, or 
xe2x80x83wherein
B represents CH2, Cxe2x95x90O or SO2,
D represents a radical selected from the following group: 
xe2x80x83In the definition for the substituent D,
m denotes an integer of 0 to 1,
n denotes an integer of 1 to 2,
X represents hydrogen,
R1 and R2 independently of one another represent hydrogen or lower alkyl,
E represents hydrogen, phenyl, naphthyl, or 
xe2x80x83wherein
R3 and R4 independently of one another represent hydrogen, lower alkyl, or 2-methoxyethyl,
G represents a radical selected from the following group: 
xe2x80x83wherein
Z represents O or Nxe2x80x94R6 (wherein R6 represents methyl),
R7 and R8 independently of one another represent hydrogen.
Typical examples of the compound of formula (1) according to the present invention are presented in the following Table 1.
It is another object of the present invention to provide processes for preparing the piperidine derivative of formula (1) as defined above.
According to the present invention, the piperidine derivative of formula (1) can be prepared by a process characterized in that
(a) a compound represented by the following formula (2a): 
wherein Cbz represents benzyloxycarbonyl and has the same meaning through the present specification, is reacted in a solvent in the presence of a base with a compound represented by the following formula (3): 
wherein E and G are defined as previously described, then the protecting group Cbz is eliminated to produce a compound represented by the following formula (1a): 
wherein E and G are defined as previously described;
(b) the compound of formula (1a) is reacted in a solvent with a compound represented by the following formula (4):
Axe2x80x2xe2x80x94Wxe2x80x83xe2x80x83(4)
wherein Axe2x80x2 is the same with A except that Axe2x80x2 is not hydrogen, and W represents hydrogen, hydroxy or reactive leaving group, preferably halogen, to produce a compound represented by the following formula (1b): 
wherein Axe2x80x2, E and G are defined as previously described;
(c) the compound of formula (1a) is reacted in a solvent with a compound represented by the following formula (5):
Axe2x80x3xe2x80x94Nxe2x95x90Cxe2x95x90Oxe2x80x83xe2x80x83(5)
wherein Axe2x80x3 represents lower alkyl, benzyl or C3-C6-cycloalkyl, to produce a compound represented by the following formula (1c): 
wherein Axe2x80x3, E and G are defined as previously described;
(d) the compound of formula (1a) is reacted in a solvent in the presence of a reducing agent with a compound represented by the following formula (6):
Dxe2x80x94CHOxe2x80x83xe2x80x83(6)
wherein D is defined as previously described, to produce a compound represented by the following formula (1d): 
wherein D, E and G are defined as previously described; or
(e) the compound of formula (1a) is reacted in a solvent with phosgene and a compound represented by the following formula (7):
xe2x80x83Dxe2x80x2Hxe2x80x83xe2x80x83(7)
wherein Dxe2x80x2 represents a radical selected from the following group: 
wherein m, n, X, R1 and R2 are defined as previously described, to produce a compound represented by the following formula (1e): 
wherein Dxe2x80x2, E and G are defined as previously described.
However, the compound according to the present invention may be conveniently prepared by any methods designed by combining various synthetic ways known in the prior arts, and such combination can be easily performed by a person having ordinary skill in this art. The process variants (a) to (e) will be more specifically explained below.
In process variants (a) to (e) for preparing the compound according to the present invention, any inert solvent which does not adversely affect to the reaction, preferably one or more selected from a group consisting of dimethylformamide, dichloromethane, tetrahydrofuran, chloroform and dimethylacetamide can be used. As the base in process variant (a), one or more selected from a group consisting of sodium hydride, potassium t-butoxide, sodium bis(trimethylsilyl)amide and potassium bis(trimethylsilyl)amide can be mentioned. Also, the deprotection reaction in process (a) to remove the benzyloxycarbonyl group at position-1 of piperidine ring may be carried out by applying the conventional reaction conditions, preferably by using Pd(OH)2/C or Pd/C in an alcohol solvent under hydrogen atmosphere.
In process variant (b), the compound of formula (1a) obtained in process variant (a) is coupled with the compound of formula (4) in the solvent as mentioned above optionally in the presence of a tertiary amine base to produce the compound of formula (1b). When the compound of formula (4) wherein W is hydroxy is used, the reaction is preferably carried out in the presence of a coupling agent. As the coupling agent, a mixture of 1-hydroxybenzotrizole(HOBT) and one or more substances selected from a group consisting of carbodiimides such as dicyclohexylcarbodiimide(DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC), 1,1xe2x80x2-dicarbonyldiimidazole(CDI), etc. can be mentioned.
The compound of formula (1) wherein B is Cxe2x95x90O, D is lower alkyl, benzyl or amino substituted by C3-C6-cycloalkyl[=compound of formula (1c)] may be prepared by reacting the compound of formula (1a) obtained in process variant (a) with the isocyanate derivative of formula (5).
In process variant (d), a reductive amination reaction is carried out in the presence of a reducing agent. As the reducing agent which can be used in this reaction, those conventionally recognized as a weak reducing agent such as Pd/C under hydrogen atmosphere, sodium triacetoxyborohydride or sodium cyanoborohydride can be mentioned.
On the other hand, a compound represented by the following formula (2) which includes the compound of formula (2a) used as a starting material in process variant (a) is a novel compound. Therefore, it is another object of the present invention to provide the compound of formula (2): 
wherein Y represents hydroxy or chloro.
The novel intermediate of formula (2) can be prepared by processes characterized in that
(f) a compound represented by the following formula (8): 
xe2x80x83is desulfurated in an organic solvent in the presence of nitric acid to produce a compound represented by the following formula (2b): 
xe2x80x83or
(g) the compound of formula (2b) is reacted with thionyl chloride(SOCl2) to produce the compound of formula (2a): 
In order to desulfurate the compound of formula (8), 10% nitric acid is used in the present invention. At this time, a small quantity of organic solvent should be added to the reaction solution because of the solubility problem of the bulky benzyloxycarbonylpiperidine group. Ethyl acetate or tetrahydrofuran can be used as the organic solvent. However, it is also possible to prepare the compound of formula (2b) from the compound of formula (8) using the other processes known as desulfuration conditions. In addition, the compound of formula (2b) thus obtained may be reacted with thionyl chloride to effectively prepare the compound of formula (2a).
As depicted in Reaction Scheme 1 below, the compound of formula (8) used as a starting material in preparing the compound of formula (2) may be synthesized from 4-(aminomethyl)piperidine by a process, in which protection, benzyloxycarbonylation and deprotection to an amine compound, and then reaction with dihydroxyacetone in the presence of KSCN are included. J. Med. Chem., 33, 1312-1329, 1990 in which a similar reaction is explained in detail can be referred to for the specific reaction conditions. 
in the above Reaction Scheme 1
CbzCl represents benzylchloroformate and has the same meaning through the present specification.
The compound of formula (3) used as a reactant in preparing the compound of formula (1) may be synthesized from 1-naphthaldehyde or 1-naphthoic acid as depicted in Reaction Scheme 2 below. 
in the above Reaction Scheme 2
TosMIC represents tosylmethylisocyanide and has the same meaning through the present specification.
The solvents which can be used in the first and second steps of Reaction Scheme 2 above include tetrahydrofuran, acetonitrile and dimethylformamide. As the base, one or more selected from a group consisting of potassium t-butoxide, 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU), potassium hydroxide and sodium hydroxide can be mentioned.
The reaction conditions including the amount of reactants, reaction temperature, reaction time, etc. in the processes according to the present invention can easily be determined by a person skilled in this art depending on the specific reactants.
In addition, the compound of formula (1) produced in the above processes in the form of a free base can easily be converted to a salt form as mentioned above according to the conventional methods known in this art.
After the reaction is completed, the resulting product may be further separated and/or purified by usual work-up processes, such as for example, chromatography, recrystallization, etc.
The compound of formula (1) prepared according to the processes above shows an inhibitory activity against farnesyl transferase, and thus can be effectively used as an anti-cancer agent. Therefore, the present invention also provides a pharmaceutical composition comprising the novel compound of formula (1), as defined above, or a pharmaceutically acceptable salt thereof as an active ingredient together with a pharmaceutically acceptable camer. Particularly, the compound of formula (1) can be used very effectively for treating cancer, restenosis, atherosclerosis and infections from hepatitis delta and related viruses.
When the active compound according to the present invention is used for clinical purpose, it is preferably administered in an amount ranging from 5 to 200 mg per kg of body weight a day. The total daily dosage may be administered in one time or over several times. However, the specific administration dosage for the patient can be varied with the specific compound used, body weight of the subject patient, sex, hygienic condition, diet, time or method of administration, excretion rate, mixing ratio of the agent, severity of the disease to be treated, etc.
The compound of the present invention may be administered in the form of injections or oral preparations. Injections, for example, sterilized aqueous or oily suspension for injection, can be prepared according to the known procedure using suitable dispersing agent, wetting agent, or suspending agent. Solvents which can be used for preparing injections include water, Ringer""s fluid and isotonic NaCl solution, and also sterilized fixing oil may be conveniently used as the solvent or suspending media. Any non-stimulative fixing oil including mono-, di-glyceride may be used for this purpose. Fatty acid such as oleic acid may also be used for injections.
As the solid preparation for oral administration, capsules, tablets, pills, powders and granules, etc., preferably capsules and tablets can be mentioned. It is also desirable for tablets and pills to be formulated into enteric-coated preparation. The solid preparations may be prepared by mixing the active compound of formula (1) according to the present invention with at least one carrier selected from a group consisting of inactive diluents such as sucrose, lactose, starch, etc., lubricants such as magnesium stearate, disintegrating agent and binding agent.